Antigens and antigen combinations

Abstract

NTHI protein antigens have been identified and found to be conserved across several Haemophilus influenzae pathogenic strains. They have been isolated, cloned from a reference strain and tested for immunogenicity. Methods for immunization and vaccines derived thereof are also disclosed.

Claims

1. An immunogenic composition comprising: an effective amount of an adjuvant; at least one isolated or recombinant polypeptide antigen from non-typeable Haemophilus influenza selected from a group consisting of: NTHI1292 (NT067) and NT113, and at least one isolated or recombinant polypeptide antigen from non-typeable Haemophilus influenza selected from the group consisting of: NTHI0267 (NT035) and NTHI0409 (NT025).

2. The composition of claim 1 wherein, said NT067 antigen is a polypeptide that comprises an amino acid sequence: (a) having 80% or more identity to SEQ ID NO: 5 or to SEQ ID NO: 52; and/or (b) that is a fragment of at least 10 consecutive amino acids, and comprises an epitope, of SEQ ID NO: 5 or SEQ ID NO: 52.

3. The immunogenic composition according to claim 1, further comprising at least one polypeptide selected from the group consisting of: (24) P48 (NTHI0254 also defined as NT007), (25) HtrA (NTHI1905 also defined as NT006), (26) PE (NTHI0267 also defined as NT035), (27) P26 (NTHI0501 also defined as NT010), (28) PHiD (NTHI0811 also defined as NT080), and (29) P6 (NTHI0501, also defined as NT081).

4. The composition of claim 1, further comprising at least one vaccine antigen that is not a non-typeable H. influenzae antigen selected from the group consisting of: an antigen from N. meningitidis serogroup A, B, C, W135, or Y, or a combination thereof; a saccharide or polypeptide antigen from Streptococcus pneumonia; an antigen from hepatitis A virus; an antigen from hepatitis B virus; a diphtheria antigen; a tetanus antigen; an antigen from Bordetella pertussis; a whole cellular pertussis antigen; a saccharide antigen from Haemophilus influenzae B; a polio antigen; a measles, mumps, or rubella antigen, or a combination thereof; an influenza antigen; an antigen from Moraxella catarrhalis; an antigen from Respiratory Syncytial Virus; a vaccine composition comprising diphtheria (D), tetanus (T), pertussis (acellular, component) (Pa), hepatitis B (rDNA) (HBV), poliomyelitis (inactivated) (IPV) and Haemophilus influenzae type b (Hib) conjugate vaccine (adsorbed); and, a combination of any of the foregoing.

5. The composition of claim 1 which further comprises one or more pharmaceutically acceptable carriers, diluents, or a combination thereof.

6. A vaccine comprising the composition of claim 1.

7. A method for raising an immune response against a non-typeable H influenza, comprising the step of administering to a mammal an effective amount of the composition of claim 1.

8. A method for raising an immune response against a non-typeable H influenza, comprising the step of administering to a mammal an effective amount of the composition of claim 3.

9. The immunogenic composition according to claim 1, further comprising at least one isolated or recombinant polypeptide antigen from non-typeable Haemophilus influenza, selected from a group consisting of: (8) CGSHiGG_00130 (NT052), (1) NTHI_0915 (NT018), (2) NTHI1416 (NT024), (3) NTHI12017 (NT032), (4) CGSHiGG 02400 (NT038), (6) NTHI0877 (NT001), (7) NTHI0266 (NT016), (9) NTHI1627 (NT002), (10) NTHI1109 (NT026), (11) NTHI0821 (NT009), (12) NTHI0409 (NT025), (13) NTHI1954 (NT028), (14) NTHI0371 (NT029), (15) NTHI0509 (NT031), (16) NTHI0449 (NT015), (17) NTHI1473 (NT023), (18) gi-145633184 (NT100), (19) NTHI1110 (NT040), (20) gi-46129075 (NT048), (21) gi-145628236 (NT053), (22) NTHI1230 (NT066), (23) NTHI0522 (NT097), (24) NT004, (25) NT014, (26) NT022, (30) NT013, (31) NT106, (32) NT107, (33) NT108, (34) NT109, (35) NT110, (36) NT111, (37) NT112, (39) NT114, (40) NT115, (41) NT116, (42) NT117, (43) NT118, (44) NT123, (45) NT124, (46) NT119, (47) NT120, (48) NT121, (49) NT122, and (50) NT061.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a mini-induction confirming strong expression of the antigens in BL21 (DE3)T1.sup.r cells. (a): (LMWM: molecular weight standard markers)

(2) FIG. 2 shows various results for NT001, NT007, NT018, NT024, NT032 and NT067. Similar expression results were obtained with the other preferred antigens, such as NT052, NT004, NT014, NT016 or NT022. Each panel shows western blot and FACS data. The western blots were performed using mouse sera, and lanes show reactivity with total bacterial extracts (TE), with vesicles prepared from NTHI outer membranes (OMV), or with purified recombination protein (PP). The FACS analyses follow incubation of inactivated bacteria with sera from mice immunized with various antigen compositions using only alum as negative control; pre-immune serum negative controls are shown as solid areas, and surface expression signal obtained with sample serum is shown as a single line.

(3) FIG. 3 shows the layout on a 96 well plate of a serum bactericidal assay to verify the capacity of antisera against antigens of the invention to kill NTHI.

MODES FOR CARRYING OUT THE INVENTION

(4) Overview

(5) Antigens list all of them which were identified as conserved in a comparative analysis performed by the inventors of at least 86 different NTHI strains, were cloned and expressed. The proteins were purified and used to immunize mice. Antisera from the immunized mice were used to verify surface localization and protective capability of the proteins used in immunization (Table III and/or Table IV). The results show that immunization NT052, NT024, NT032, NT001, NT067, NT004, NT014, NT022, NT016 is highly protective against NTHI and they showed higher or at least comparable bacterial killing activity SBA (Serum bactericidal assay) titers even compared with the second antigen group.

(6) Strains and Variants

(7) Inventors found that genes encoding NT022, NT016, NT014, NT018, NT024, NT032, NT067 and NT001 were present and conserved in all 86 genome sequences analysed.

(8) The encoded NT018 sequences were 95-100% identical across the panel composed by the 15 complete genomes and the 32 strains from the Finnish otitis collection. The encoded NT024 sequences were 90-100% identical in the panel composed by the 15 complete genomes and the 32 strains from the Finnish otitis collection.

(9) The encoded NT032 sequences were 95-100% identical in the panel composed by the 15 complete genomes and the 32 strains from the Finnish otitis collection; the encoded NT067 sequences were 95-100% identical in the panel composed by the 15 complete genomes and the 32 strains from the Finnish otitis collection. The encoded NT001 sequences were 95-100% identical in the panel composed by the 15 complete genomes and the 32 strains from the Finnish otitis collection.

(10) Conservation in the encoded amino acid sequences are shown in Table I.

(11) TABLE-US-00001 TABLE I antigen conservation (% identity) amongst Haemophilus genomes and strains Antigen NT018 NT024 NT032 NT067 NT001 % 95-100 90-100 95-100 95-100 95-100

(12) For expression purposes, antigens belonging to the first antigen group and/or second antigen group were cloned from either strain Fi176 which is one strain isolated from a Finnish collection of strains obtained from patients with otitis media or from strain R2846 [146]. Most of the antigen selected and further tested in animal model are also found to be well conserved amongst strains, e.g. NT016, NT067, NT022, NT014.

(13) In some cases mutations have been introduced into the wild-type sequences. These mutations are underlined in the sequence listing for NT018, NT067, NT001, NT016, NT002, NT026, NT009, NT015, NT023 and NT066 (see SEQ ID NOs: 49, 52, 54, 55, 57-59, 64, 65 & 67).

(14) Cloning and Expression of NTHI Recombinant Proteins

(15) Cloning and expression of antigens can be performed by standard methods [121].

(16) ORFs for antigens from NTHI strain Fi176 or R2846 were PCR-amplified using specific oligonucleotides and NTHI chromosomal DNA as template. Resulting PCR products were cloned in pET15b (Novagen) using the PIPE method [147], consisting in the PCR amplification of the cloning vector (V-PCR) and in the39 PCR amplification of the insert (I-PCR). Then, 1 l of V-PCR and 1 l of I-PCR are mixed and transformed in chemically competent HK100 cells [148]. I-PCR reactions were set up containing 1 M each of the forward and reverse primers, 1 Cloned Pfu DNA Polymerase Reaction Buffer, 2.5 units of Pfu Turbo DNA polymerase (Stratagene), 200 M of each dNTP (Invitrogen) and 50 ng of genomic DNA template. The reactions were conducted as follows: initial denaturation for 2 min at 95 C., then 25 cycles of 95 C. for 30 s, 55 C. for 45 s, and 68 C. for 3 min followed by a final cool down to 4 C. V-PCR reactions were identical to the I-PCR reactions but the steps at 68 C. were lasting 14 min and 2 ng of pET15b plasmid were used as DNA template. Correct transformants where selected by PCR screening and DNA plasmid sequencing of the vector-insert junctions. The correct plasmid were then prepared from selected HK100 clones and used to transform BL21(DE3)T1.sup.r cells (Sigma) in order to allow protein expression.

(17) To express cloned proteins, BL21(DE3)T1.sup.r clones containing pET15b constructs were grown in LB medium containing 100 g/ml Ampicilin at 37 C. until OD.sub.600=0.5. Protein expression was then induced by adding 1 mM IPTG and growing at the same temperature for additional 3 hrs. Conventional protein extractions and SDS-Page were performed to check protein expression. FIG. 1 shows a mini-induction confirming good expression of the antigens.

(18) Protein Purification

(19) Proteins were purified by the following general procedure: BL21(DE3)T1 wet biomass is suspended in lysis buffer and clarified by centrifugation. For purification of soluble protein ( ) supernatants after lysis are applied on His Multitrap HP 50 l NiSepharose High Performance 96 well plates. For insoluble protein (HtrA, PE and P48), pellets containing the unsoluble fraction after lysis are solubilised with 6M Guanidine-HCl and re-centrifuged, and the supernatants applied to His Multitrap HP 50 ml NiSepharose High Performance 96 well plates.

(20) Flow-through is collected and all wells washed with buffer containing 20 mM imidazole. His fusion proteins are then eluted with 250 mM imidazole. The procedure is performed using a vacuum system. Purified antigens are used in the immunisation schemes described herein.

(21) The following protocol was followed: 1) Resuspend BL21(DE3)T1 pellet (1 g) in 1.5 ml B-PER (PIERCE) buffer, add 15 l of lysozyme, 7.5 l DNAse and 3 l of MgCl.sub.2 1 M 2) Incubate for 30 min for lysis 3) Centrifuge at 20000 rpm at 4 C. for 30 minute; for purification of any insoluble protein, solubilise pellets containing the unsoluble fraction after lysis with 6M Guanidine-HCl and re-centrifuge 4) Recover supernatant and filter (pore of 0.8 m). 5) Use His Multitrap HP 50 l NiSepharose High Performance 96wells, connected to a vacuum system

(22) Buffer A: 50 mM NaPPi, 300 mM NaCl, pH8

(23) Buffer B: 50 mM NaPPi, 300 mM NaCl, 250 mM Imidazole, pH8

(24) Buffer C: 50 mM NaPPi, 300 mM NaCl, 20 mM Imidazole, pH8

(25) 1st Step: remove ethanol from the plate.

(26) 2nd Step: wash the plate with 400 l of milliQ H2O.

(27) 3rd Step: equilibrate the plate with 400 l A of Buffer A

(28) 4th Step: load 600 l of starting material for each protein in one of the 12 columns. If the volume is larger, repeat until all the material is fully loaded.

(29) Recover the flow through.

(30) 5th Step: Wash Step: 4 washes with 400 l of Buffer C. Discard the flow through.

(31) 6th Step: Elution: 2300 l Buffer B (2 elution steps).

(32) Activate vacuum 15 minutes after adding the buffer.

(33) 1 l of total extract, 1 l of starting material, 1 l of flow through and 10 l of elution volume (for each protein) are analysed by SDS-PAGE.

(34) For insoluble protein, buffer B is replaced by 10 mM tris, 50 mM Na.sub.2HPO.sub.4, 8 M urea, 250 mM imidazole, 40% glycerol.

(35) LAL Test

(36) The LAL test is a test that measures the endotoxin concentration in a vaccine sample using the endosafe-PTS Charles River technology.

(37) Test Technology

(38) The PTS utilizes LAL kinetic chromogenic methodology to measure color intensity directly related to the endotoxin concentration in a sample. Each cartridge contains precise amounts of licensed LAL reagent, chromogenic substrate, and control standard endotoxin (CSE). The cartridges are manufactured according to rigid quality control procedures to ensure test accuracy and product stability.

(39) TABLE-US-00002 TABLE II Purification of preferred antigens Purity % LAL Internal kDa kDa RP- SE- SE- Test ID Annotation (expected) (SE estimated) Soluble densitometry HPLC UPLC HPLC EU/g nt001 NTHI0877 30 36 yes 97 84 monomer 0.47 nt018 NTHI0915 34 46 yes 80 88 monomer 0.18 nt024 NTHI1416 20 20 yes 81 85 97 monomer 3.77 nt032 NTHI2017 13 16 yes 91 76 monomer 0.82 nt067 NTHI1292 60 50 yes 88 78 monomer 0.06 nt052 CGSHiGG_00130 34 46 yes 88 88 monomer 0.18 nt004 CGSHiGG_08215 20 34 yes 95 95 monomer 0.09 nt014 HI1658 20 17 yes 89 87 monomer 0.10 nt022 NTHI0830 43 77 yes 93 93 monomer 0.10 nt016 NTHI0266 29 30 yes 98% monomer 0.13

(40) Immunisation of Mice and Production of Antisera

(41) Five weeks old CD1 mice (8 for each antigen) were immunized by 3 intraperitoneal injections (every two weeks) of 10 micrograms of purified protein antigens with Freund's adjuvant (200 microliters per mouse) or with Alum (aluminium hydroxide adjuvant; 2 mg/ml). Sera were collected two weeks after the third injection and stocked at 20 C. Controls were injected with Freund's adjuvant only or alum only.

(42) FACS Analysis

(43) A surface labeling assay by FACS was performed in order to examine the surface exposure of the selected antigens and the levels of expression in different strains. NTHI were incubated with sera derived from mice immunized with recombinant proteins or negative controls, and analysed by FACS. The results are shown in FIG. 2. In FIG. 2, pre-immune serum negative controls are shown as solid areas, and the signal obtained with sample serum is shown as a single line. The results of FACS analyses of antigens P48, HtrA, PE, and P26 demonstrate that each of these antigens is exposed on the surface of the bacterium and thus accessible to antibody binding.

(44) The following materials and methods were used in this analysis:

(45) Materials

(46) 1. 96 U-bottom well plates. 2. Blocking and Washing Buffer: PBS containing 1% (w/v) BSA. 3. Goat anti-mouse IgG-Fluorescein IsoThio Cyanate FITC. 4. PBS containing 0.5% (v/v) para-formaldehyde: dilute a stock solution of 4% (v/v) para-formaldehyde in PBS to 0.5% (v/v) fresh before the assay and filter sterilize (0.22 m filter). 5. PBS containing 1% (w/v) BSA. To prepare this solution, dissolve 1% (w/v) BSA in PBS, making at least 100 ml for each strain. Filter-sterilize the solution (0.22 m filter) and prepare fresh for use. 6. FACScan tubes (Becton Dickson). 7. FACScalibur flow cytometer (Becton Dickinson).
Methods 1. Grow NTHI until an OD.sub.600 nm value of 0.5 is reached, then transfer 1 ml of culture to a sterile 1.5 ml Eppendorf tube and centrifuge at 13000 g in a micro-centrifuge for 3 minutes to pellet the bacteria. Discard the supernatant and suspend the pellet suspended in 1 ml of PBS containing 1% (w/v) BSA. Finally, dilute the bacterial suspension 1/50 in PBS containing 1% (w/v) BSA. 2. Add 50 l samples of sera diluted in Blocking Buffer (at 1/100, 1/200 and 1/400) in a 96 well plate. Include positive controls, such as anti-OMV antisera, 3. Add 50 l of bacterial cells to each well and store the plate at 4 C for 2 h. 4. Centrifuge the cells for 5 minutes at 3500 g, discard the supernatant and wash the cells by adding 200 l/well of Washing Buffer. 5. Add 50 l of a 1/100 dilution of FITC-conjugated goat anti-mouse Ig to each well and store the plate at 4 C. for 1 h. 6. Centrifuge the cells at 3500 g for 5 min and wash the pellet with 200 l/well of PBS. 7. Repeat the centrifugation step, discard the supernatant and add 200 l/well of PBS containing 0.5% (v/v) para-formaldehyde, in order to fix the cells. 8. Transfer the fixed samples to individual FACScan tubes and analyse by flow cytometry, following the equipment manufacturer's instructions.
Serum Bactericidal Assay (SBA)

(47) Antisera derived from mice immunized with recombinant proteins were tested in a serum bactericidal assay, to verify the presence of functional antibodies able to induce killing of NTHI. Pre-immune sera and sera from mice injected only with adjuvant were used as negative controls. NTHI (strain 176) culture (BHI+NAD and Haemin) was incubated at 37 C. with shaking, until OD595 nm was 0.25-0.27. The bacterial cells were diluted in D-PBS buffer at the working dilution 1:50000. Sera were inactivated at 56 for 30 minutes and then serially diluted in D-PBS in a 96-well U-bottom plate (see FIG. 3). Columns 11 and 12 of the plate shown in FIG. 3 contain negative controls to assess the growth of the bacteria and to detect any non-complement mediated killing. Bacteria and a source of complement (Rabbit 7504, Cedarlane) were added to each well except in the complement control wells which received heat-inactivated complement.

(48) As shown in FIG. 3, wells in columns 1-10 contain 25 l diluted sera, 12.5 l active complement, and 12.5 l bacteria. Wells in column 11 contain 25 l buffer, 12.5 l active complement, and 12.5 l bacteria. Wells in column 12 contain 25 l buffer, 5 l sera, 12.5 l heat inactivated complement, and 12.5 l bacteria.

(49) 10 l of the time zero (TO) assay controls (column 11-12) were plated on agar chocolate plate (Biomerieux) by the spot and tilt method. Plates were incubated at 37 C., ON. The assay microtiter plates were incubated for 1 hour at 37 C. After this period (T60) 7 l of each well were plated as spot on an agar chocolate plate (each well was plated in duplicate). The number of colonies (colony forming units, CFU) was counted using a colony counter or manually. A bactericidal effect was considered to be observed when the number of colonies was lower than 50% of T=0.

(50) An overview of the results is provided in the following Table III and Table IV.

(51) TABLE-US-00003 TABLE III Immunogenicity results SBA TITER Freund's Internal ID Annotation SEq ID NOs kDa adjuvant (176 wt) FACS NT001 NTHI0877 SEQ ID NO: 6 or 30 2048-8192 +++++ SEQ ID NO: 54 NT016 NTHI0266 SEQ ID NO: 7 or 29 2048-8192 +++++ SEQ ID NO: 55 NT024 NTHI1416 SEQ ID NO: 2 or 20 2048-8192 ++ SEQ ID NO: 50 NT032 NTHI2017 SEQ ID NO: 3 or 13 2048-8192 + SEQ ID NO: 51 NT018 NTHI0915 SEQ ID NO: 1 or 34 2048-4096 + SEQ ID NO: 49 NT038 CGSHiGG_02400 SEQ ID NO: 4 or 22 2048-4096 ++ SEQ ID NO: 50 NT052 CGSHiGG_00130 SEQ ID NO: 8 or 44 2048-4096 ++ SEQ ID NO: 56 NT067 NTHI1292 SEQ ID NO: 5 or 60 2048 ++ SEQ ID NO: 52 NT002 NTHI1627 SEQ ID NO: 9 or 18 1024-2048 +++ SEQ ID NO: 57 NT026 NTHI1109 SEQ ID NO: 10 or 19 4096-8192 ++++ SEQ ID NO: 58 NT009 NTHI0821 SEQ ID NO: 11 64 4096 +++ or SEQ ID NO: 59 NT025 NTHI0409 SEQ ID NO: 12 17 4096 ++++ or SEQ ID NO: 60 NT028 NTHI1954 SEQ ID NO: 13 20 4096 +++ SEQ ID NO: 61 NT029 NTHI0371 SEQ ID NO: 14 101 4096 +++ SEQ ID NO: 62 NT031 NTHI0509 SEQ ID NO: 15 20 4096 + SEQ ID NO: 63 NT015 NTHI0449 SEQ ID NO: 16 15 2048-4096 ++ SEQ ID NO: 64 NT023 NTHI1473 SEQ ID NO: 17 17 2048-4096 ++ SEQ ID NO: 65 NT100 gi145633184 SEQ ID NO: 18 34 2048-4096 + SEQ ID NO: 66 NT040 NTHI1110 SEQ ID NO: 19 26 1024-2048 + NT048 gi-46129075 SEQ ID NO: 20 71 1024-2048 + NT053 gi145628236 SEQ ID NO: 21 17 1024-2048 + NT066 NTHI1230 SEQ ID NO: 22 59 1024-2048 + SEQ ID NO: 67 NT097 NTHI0522 SEQ ID NO: 23 50 1024-2048 ++ NT006 NTHI1905 SEQ ID NO: 25 51 2048 ++++ (HtrA) NT035 (PE) NTHI0267 SEQ ID NO: 26 18 512-1024 ++ NT080 NTHI0811 SEQ ID NO: 28 512 (PHiD) NT081 (P6) NTHI0501 SEQ ID NO: 29 512 NT010 (P26) NTHI1083 SEQ ID NO: 27 22 128-512 +++ NT007 (P48) NTHI0254 SEQ ID NO: 24 48 8192-16384 +++++ Unrelated 16 + antigen Freund's 512/1024 Adj. alone

(52) These results show that antigens selected are highly effective in killing NTHI pathogens. In particular NT018, NT001, NT024, NT032, NT067, NT016 all show particularly strong protective effects.

(53) TABLE-US-00004 TABLE IV Immunization experiments using compositions comprising NTHI antigens and Alum Protein Pu- SBA SBA FACS FACS Solu- Antigen rity (Freund) (Alum) Freund alum bility NT001 97% 2048-8192 512-1024 +++++ ++ + Yes NT024 94% 2048-8192 1024 ++ ++ + Yes NT038 97% 2048-4096 64 ++ Yes NT018 80% 2048-4096 512-1024 + +++ Yes NT032 99% 2048-8192 64-128 + + Yes NT067 88% 2048 512-2048 ++ +++ Yes NT025 94% 4096 128-256 ++++ + No NT026 64% 4096-8192 64 ++++ No NT028 81% 4096 128-256 +++ ++ No NT029 52% 4096 128 +++ ++ Yes NT023 80% 2048-4096 256-512 ++ + Yes NT015 78% 2048-4096 128-256 ++ + No NT031 90% 4096 128 + + No NT100 81% 2048-4096 512 + + Yes NT081 (P6) 88% 2048 256 + + No NT080 92% 1024 128 + + (PHiD) NT006 57% 2048 256 ++++ ++++ (HtrA) NT007 (P48) 79% 8192-16384 256-512 +++++ +++++ NT052 88% 2048-4096 512-1024 + +++ Yes NT014 87% 1024 512-1024 ++ ++ Yes NT004 95% 256-512 128-256 ++ + Yes NT022 93% 64-256 1024 +++ + Yes NT016 98% 2048-8192 128 +++ ++++ Yes NT106 82% Not tested 64-128 ++ +++ Yes NT113 92% Not tested 128 ++ +++ Yes NT061 83% Not tested 128 +++ +++ Yes Freund's 512 NA Alum NA 4-8

(54) These results further confirmed that antigens selected are highly effective in killing NTHI pathogens also when used in immunogenic compositions with alum as adjuvant.

(55) In particular NT016, NT052, NT018, NT001, NT024, NT032, NT067, NT014, NT022 all confirm particularly strong protective effects as measured in serum bactericidal assay (SBA).

(56) Particularly preferred antigens were NT067, NT014, NT016, NT022. These antigens have been also tested in an in vivo animal model according to the protocol described in Ref. 75.

(57) In vivo Vaccine Efficacy Testing

(58) Individual antigens as listed in Table IV can be tested for their ability to protect against an otitis media (OM) infection using an in vivo model such as Junbo and Jeff mouse mutants [75].

(59) The vaccine efficacy in the in vivo protection experiment is performed using 3 administrations (at day 0, 21, 35) of 10 micrograms/mouse of purified recombinant protein antigens formulated with or without adjuvant, followed by intranasal inoculation with selected NTHI pathogenic strains.

(60) 10 Pre-immune sera, post-immunization sera, and terminal sera 7 days post-NTHI inoculation are collected and stored at 80 C. Controls are immunized with adjuvant or with an unrelated antigen as control. Middle ear bulla and nasopharyngeal (NP) washes samples are collected and plated to determine NTHi numbers; bulla infection and nasopharingeal carriage rates, and bulla NTHi titres are then calculated.

(61) It will be understood that the invention is described above by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

(62) TABLE-US-00005 TABLE V Nomenclature cross-reference with representative strains 86-028NP SEQ ID NOs Name NTHI_# 3655 Strain PittG Strain 1 or 49 NT018 NTHI0915 2 or 50 NT024 NTHI1416 3 or 51 NT032 NTHI2017 4 or 53 NT038 CGSHiGG_02400 5 or 52 NT067 NTHI1292 6 or 54 NT001 NTHI0877 7 or 55 NT016 NTHI0266 8 or 56 NT052 CGSHiGG_00130 9 or 57 NT002 NTHI1627 10 or 58 NT026 NTHI1109 11 or 59 NT009 NTHI0821 12 or 60 NT025 NTHI0409 13 or 61 NT028 NTHI1954 14 or 62 NT029 NTHI0371 15 or 63 NT031 NTHI0509 16 or 64 NT015 NTHI0449 17 or 65 NT023 NTHI1473 18 or 66 NT100 gi-145633184 19 NT040 NTHI1110 20 NT048 gi-46129075 21 NT053 gi-145628236 22 or 67 NT066 NTHI1230 23 NT097 NTHI0522 24 NT007 P48 25 NT006 HtrA 26 NT035 PE 27 NT010 P26 28 NT080 PHiD 29 NT081 P6 30 NT013 NTHI0532 31 NT106 NTHI0363 32 NT107 NTHI0370 33 NT108 NTHI0205 34 NT109 NTHI0374 35 NT110 NTHI0579 36 NT111 NTHI0837 37 NT112 NTHI0849 38 NT113 NTHI0921 39 NT114 NTHI0995 40 NT115 NTHI1091 41 NT116 NTHI1169 42 NT117 NTHI1208 43 NT118 NTHI1318 44 NT123 NTHI1796 45 NT124 NTHI1930 114 NT119 NTHI1565 115 NT120 NTHI1569 116 NT121 NTHI1571 117 NT122 NTHI1667 122 NT004 CGSHiGG_08215 123 NT014 gi-145629254 124 NT022 NTHI0830 128 NT061 NTHI0588 130 NT017 NTHI0915

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